A major goal of genome sequencing efforts is to enable patient-specific therapies. Our laboratory is focused on developing general approaches to design selective lead therapeutics from only genome sequence. Although oligonucleotides have been used to target and study RNA, they are hampered by suboptimal properties such as poor cell and tissue permeability, limited available modifications/derivatives for optimization, an ineffective targeting of structured RNAs. We are tackling this difficult problem by targeting the RNA products of genes with small molecules, using several novel and transformative technologies we developed. These approaches uncovered new biological roles for RNA (4) and provided transformative approaches to allow for a disease-affected cell to synthesize its own drug.(9) Furthermore, we have shown that genome sequence can be utilized to afford patient-specific therapies and chemical probes of function that are highly selective - in fact with greater or equal selectivity as recognition of RNA via oligonucleotides.(1, 24) Designing small molecules that modulate RNA function is difficult in large part due to an incomplete understanding of RNA-small molecule recognition events. That is, the chemical space that is privileged for binding RNA and RNA scaffolds that form small molecule binding sites are unknown. We are filling these knowledge gaps in innovative and novel ways to enable design of chemical probes that target RNA with unprecedented accuracy. Our proposed work seeks to expand our bottom-up approach, named Inforna: Specific Aim 1: Identify drug-like scaffolds that bind RNA. To advance Inforna, two knowledge gaps need to be filled: (i) identification of highly prevalent motif types in cellular RNAs; and (ii) identification of features in drug-like small molecules that confer avidity for RNA. We will fill these knowledge gaps by probing a drug-like small molecule library for binding to important RNA motifs for which there are limited data in Inforna. Specific Aim 2: Target disease-associated miRNAs using lead small molecules identified in Specific Aim 1. Inforna has provided numerous bioactive compounds against disease-associated RNAs. In our recent report in Nature Chemical Biology,(1) it enabled a target agnostic approach to discover lead compounds against miRNA precursors, affording a 44% bioactive hit rate. Specific Aim 3a: Develop precision multivalent dimers that allow for selective, potent targeting of miRNA precursors. The central hypothesis in this sub-aim is that although there are targetable RNA motifs in the transcriptome, there are far fewer RNAs that have the multiple targetable motifs separated by the same distance. Specific Aim 3b: Develop small molecules that target two miRNAs with a single small molecule and study the cellular consequences of dual targeting of disease pathways via designer poly-pharmacy.